Activation of carcinogens: the enzymes

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Activation of carcinogensthe enzymes

• Chemicals, Risk Cancer 4
• David R. Bell
• Annual review of pharmacology and toxicology
  yr2003 vol43 pg149 -173 Mutation Research 488
  (2001) 195209 Toxicology and Applied
  Pharmacology 206 (2005) 73 93

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Activation

• Activation is split into two phases
• Phase I
• Direct oxidation/ metabolism of the primary
  compound
• Phase II
• Conjugation of the compound/ metabolite with a
  hydrophilic compound (glutathione, SO4, sugar)

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Why activate ?

• Why activate compounds ?
• Hydrophobic compounds are bad
• Accumulation of hydrophobic compounds in cell
  membranes/ fatty compartments
• Accumulation means very high concentrations in
  specific organs
• Highly toxic compounds are often highly
  hydrophobic

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Fatty compounds accumulate
Fat
Blood
In
Brain
Out
Kidney
Other tissue
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Why metabolism ?

• Metabolism of foreign compounds makes them more
  hydrophilic, ie water-soluble
• Water-soluble compounds are in the bloodstream,
  and are passed by the kidneys
• Phase I- add oxygen, or hydroxy groups
• Phase II- add hydrophilic molecules to functional
  groups, e.g. OH, -NH2 created in phase I

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Metabolism

• Gut metabolism
• The gut contains a great number of bacteria
• Bacterial metabolism of foreign compounds is
  significant
• Frequently anaerobic, ie reducing
• Vs. hepatic metabolism, frequently oxidising

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Phase I

• Cytochrome P450
• Unique spectral characteristic
• Compare reduced enzyme, vs reduced enzyme CO
  gives a peak at 450 nm
• Pigment 450nm
• Has protoporphyrin IX (haem) prosthetic group
• Principally hepatic, but present in many other
  organs

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Fe3 prosthetic group
H2O
R
N
N
Fe 3
N
N
S
H
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P450 cofactors

• P450 present in the endoplasmic reticulum
• Requires
• NADPH-cytochrome P450 reductase
• (Optional) cytochrome b5
• O2
• NADPH

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P450 reaction cycle
Substrate R-H
P450 3
ROH
H2O
P450 3 -RH
NADPH P450 reductase
2 H
e-
O22-- P450 3 -RH
P450 2 -RH
P450 reductase or b5
e-
O2
O2- P450 2 -RH
O2-- P450 3 -RH
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P450 reactions

• C, N or S- hydroxylation, or oxidation
• dealkylation
• reduction
• dehalogenation
• wide variety of substrates
• multiple P450s
• each of which has wide substrate specificity

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P450 structure
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P450 gene family

• gt 1200 genes known
• Humans have 17 families, 42 subfamilies
• Present from plants, bacteria to human
• Conserved structural features
• P450s are grouped into
• Families e.g. CYP1, or CYP2
• Subfamilies, e.g. CYP1A and CYP1B
• Individual genes, e.g. CYP1A1 and CYP1A2

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P450 and cancer

• P450 4B1 (mouse, rabbit) responsible for tissue
  specific activation of aromatic amines (2AAF) in
  bladder, rabbit lung
• CYP1A1 and CYP1A2 responsible for metabolic
  activation of PAHs
• Furafylline specifically inhibits 1A2
• CYP2D6 is highly polymorphic in humans
• 95 normal metabolisers of debrisoquine
• 5 very poor metabolisers
• Relevance to cancer ?

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P450s as an adaptive response

• When yeast are grown on lipids, they induce high
  levels of fatty acid hydroxylases
• Induction of cytochrome P450 is an adaptive
  response to environmental chemicals
• The induction of cytochromes P450 1A1 in the skin
  appears to be essential for PAH carcinogenesis
• Specific inducing agents lead to the induction of
  distinct P450 forms

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Phase II

• Addition of a hydrophilic carrier moiety to make
  the compound more water soluble
• Rapid excretion
• Tissue-specific availability of Phase II enzymes
  determines further metabolism of compounds

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Sulphation

• Formation of ATP analogue, phospho-adenosine-phosp
  ho-sulphate
• The enzyme family, the sulfotransferases,
  transfer the sulphate to the substrate
• PAPS ROH 3phosphoadenosine 5 phosphate
  ROSO3H
• Widely used for the metabolism of endogenous
  substrates (steroids, neurotransmitters) and
  foreign compounds

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Acetlyation

• Addition of acetic acid in a condensation
  reaction
• R-OH CH3COOH? RO-CO-CH3 H2O
• N-acetyl transferase family is polymorphic in
  humans
• Final compound may be less hydrophilic than the
  starting compound

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Glucuronidation

• The addition of glucuronic acid, as
  UDP-glucuronic acid, catalysed by a family of
  enzymes known as UDP glucuronyl transferases
• Endogenous and exogenous substrates
• Deficiency in human UGT1.1 leads to a failure to
  excrete bilirubin, and Crigler-Najjar disease,
  characterised by a potentially fatal neurological
  syndrome (kernicterus)

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UDPGTs
COOH
COOH
UDPGT
O
O
O
OH
O-UDP
UDP
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Glutathione

• Glutathione is a tripeptide
• g-glutamyl-cys-gly
• Present in most cell types, at 1-10 mM
• Available to mop up reactive species

NH2
O
H
NH
COO-
N
-OOC
CH2
O
SH
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Glutathione S-transferase

• R-OH GSH ? R-SG H2O
• Catalysed by a family of enzymes, GSTs
• Deletion of murine GST-p cluster
• Treat mice with DMBA, wait 20 weeks
• / mice 2 papillomas/ animal
• -/- mice 9 papillomas/ animal
• GST-m is polymorphic in human

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Enzymes of metabolic activation

• Tissue-specific expression
• Species specific
• Developmental
• Sex
• Diet
• The balance between activating and detoxifying
  pathways of metabolism is determined by a complex
  interplay of metabolic pathways